Identification of disease associated antigens is an often an important step in the development of vaccines or other immune system modulators that are effective against bacterial and viral infections, cancer, autoimmune disorders, transplant rejection and bioterrorism agents. Proposed here is research to continue the development of instrumentation and methods for the sequence analysis of peptide antigens presented to the immune system in association with class I and class II molecules of the major histocompatibility complex. Routine detection and characterization of disease associated antigens present at the attomole level in a mixture of 10,000 self peptides is the goal. In the proposed research, we will implement our recently developed peak parking technology and nano-flow high performance liquid chrornatography to two new instruments, the linear trap mass spectrometer and the tandem linear trap-Fourier transform mass spectrometer. We will also continue to development the differential display software package which makes it possible to compare class I and class II peptides presented by healthy and diseased cells and then to sequence those peptides that are uniquely characteristic of the disease and likely to be recognized as antigens by the immune system. Additional aims of the proposed research include the following: (1) to identify additional class I peptides recognized by pathogenic CD8(+) T-cells in Type I diabetes, (2) to identify additional minor histocompatibility antigens associated with graft-vs-host disease and tissue transplant rejection, (3) to identify phosphorylated class I peptides presented uniquely on the surface of cancer cells, (4) to identify antigenic class I peptides presented from the prenylated breast cancer antigen, C35, (5) to identify peptide antigens restricted by class I HLA molecules on renal cell carcinoma, (6) to identify an HLA A3 restricted and shared antigen presented on melanoma cells, and (7) to evaluate the processing and mechnanism of presentation for both class I and class II epitopes from tyrosinase that are targets of T cell responses in melanoma.